The overall objective of the proposed research is to further our knowledge of the molecular mechanisms by which adenosine derivatives affect the vertebrate nervous system. During this past period of support, we found that ATP is released together with the neurotransmitter acetylcholine (ACh) within milliseconds of a nerve impulse and, after hydrolysis to adenosine, is the physiological mediator of skeletal neuromuscular depression. We also found evidence for mutually-inhibitory interactions between nicotinic ACh receptors and P2X ATP receptors in adult mammalian neurons. During the next period of support, we will continue to study the behavior of adenosine derivatives as presynaptic inhibitors of ACh release and postsynaptic inhibitors of the action of ACh on nicotinic synapses. There are two specific aims underlying the overall objective as follows. For specific aim 1, we will attempt to determine the specific molecular mechanisms responsible for the inhibitory effect of adenosine on ACh release. Towards this aim, we will activate or interfere with specific target proteins in the nerve ending and examine if the effects of adenosine are altered. For specific aim 2, we will study the mechanisms underlying the inhibitory electrophysiological interactions between nicotinic ACh receptors and P2X ATP receptors on adult mammalian sympathetic neurons. Such interactions have been found to occur even with very low concentrations of ATP and nicotine. With regards to the significance of the proposed research, adenosine derivatives have been implicated as mediators of physiological and pathological activity in the vertebrate nervous system. The experiments at the neuromuscular junction demonstrate that endogenous adenosine is a physiological mediator of neuromuscular depression at frog and mammalian synapses. Determining the specific nerve terminal targets for the inhibitory actions of adenosine would thus provide both important basic science information and potential clinical benefits. For example, it is possible that selective adenosine receptor antagonists can be used to prevent the debilitating neuromuscular depression that occurs in patients with neuromuscular disorders such a myasthenia gravis. The results with ATP will provide valuable additional knowledge on the relationship between purinergic and nicotinic synapses in mammals. Indeed, fast excitatory purinergic transmission with similar properties to the neurons we study occurs in regions of the CNS associated with cholinergic synapses and with Alzheimer's disease.